Supporting Information for:

Overexpression of GalNAc-transferase GalNAc-T3 Promotes Pancreatic Cancer Cell Growth

Supplementary Materials and Methods

Semiquantitative RT-PCR

Total RNA extracted from eight pancreatic cancer (PDAC) cell lines (PANC-1, BxPC3, S2-013, SUIT-2, COLO357, HPAF, MIA-PaCa2, and Capan2), and HPNE immortalized normal pancreatic epithelial cells were subjected to reverse transcription with StrataScript reverse transcriptase (Agilent, La Jolla, CA) and oligo d(T)12-18 primer. We prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring GAPDH as a quantitative control. The primer sequences were 5’-TGAGAACTACACGGCTGTCG-3’ and 5’-ATGGTTTGCCTCCTTGATTG-3’ forGalNAc-T3, and 5’-CGAGATCCCTCCAAAATCAA-3’ and 5’-TTCAGCTCAGGGATGACCTT-3’ forGAPDH. All reactions involved initial denaturation at 94 °C for 2 min followed by 21 cycles for GAPDH or 28 cycles for GalNAc-T3 at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min.

Immunohistochemical staining

Tissue sections from PDACs were obtained from the rapid autopsy program approved by the InstitutionalReview Board of the University of Nebraska Medical Center. Informed consent was obtained from all patients. Tissue sections from normal pancreas were purchased from Biochain (Hayward, CA). Tissue-microarray sections of PDACs (A207I, AccuMax Array) were purchased from ISU ABXIS (Seoul, Korea), where 32 PDAC tissues were spotted in duplicate. The sections were incubated with anti-GalNAc-T3 antibody followed by incubation with peroxidase-labeled anti-rabbit immunoglobulin (Envision kit; Dako Cytomation, Glostrup, Denmark). Finally, the reactants were developed with 3, 3’-diaminobenzidine (Dako) and the cells were counter-stained with hematoxylin.

Construction of small interfering RNA expressing vector and cell viability assay

The pSUPER-gfp vector (OligoEngine, Seattle, WA) was used for expression of small interfering RNA (siRNA) against GalNAcT-3. The siRNA oligonucleotide sequences for GalNAc-T3are as follows: siT3-1, 5’-GCCTGTCCTTGACCGTCCA-3’; siT3-2, 5’-GGTCTGATCACTGCTCGGT-3’; and siT3-3, 5’-GCGTTGGTCAGCCTCTATG-3’. S2-013 and BxPC3 cells were plated onto six-well plates (4  104 cells/well) and transiently transfected with empty Neo-pSUPER-gfp as a negative control or pSUPER-gfp including the target sequence for GalNAc-T3, using FuGENE6 (Roche, Penzberg, Germany), according to the manufacturer’s instructions. Cells were selected in medium containing 500 µg/ml of geneticin for 14 days and harvested after 48 h for RT-PCR analysis of the knockdown effect on GalNAcT-3. Primers for these RT-PCR experiments were the same as those described above. After 14 days of incubation in medium containing 500 µg/ml of geneticin, Giemsa staining was performed to count the number of colonies, and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cell counting kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well at a concentration of 1/10 volume, and the plates were incubated at 37 °C for an additional 3 h. Absorbance was then measured at 490 nm, and at 630 nm as a reference, with a Microplate Reader 550 (Bio-Rad, Hercules, CA).

In vitro growth rate by MTT assay

Stable GalNAc-T3 RNAi and control S2-013 cells were each seeded at a concentration of 5  104 cells per well using 12-well plates. Cell viability was examined using the cell counting kit-8 solution as described above. The MTT assay was done every 24 h for 5 days, according to the manufacturer's instructions.

Confocal immunofluorescence analysis

Cells were fixed with 4% paraformaldehyde, then permeablilized with 0.1% Triton X-100, covered with blocking solution (3% BSA/PBS), and incubated with the primary antibody for 1 h. Alexa 488-conjugated anti-rabbit IgG secondary antibody was used. Each specimen was visualized with a Zeiss LSM510 META microscope (Carl Zeiss,Gottingen, Germany).

Statistical analysis

The significance of differences between groups was determined using the Student’s t-test, Mann-Whitney U test, or Fisher’s exact test, as appropriate. P < 0.05 was considered statistically significant.

Table S1GalNAc-T3 staining in a pancreatic cancer tissue microarray

Differentiation of pancreatic tumors (n) / Strong GalNAc-T3 expression / Weak or absent GalNAc-T3 expression
Well (4) / 3 / 1
Moderate(13) / 8 / 5
Poor (9) / 6 / 3
ETC (6) / 4 / 2

ETC; Tumor cell differentiation is unknown according to the information from theISU ABXIS.

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